This invention relates to the synthesis, deprotection, and purification of RNA.
Generally, RNA molecules are chemically synthesized and purified by methodologies based on the use of tetrazole to activate the RNA phosphoramidite, ethanolic-NH4OH to remove the exocyclic amino protecting groups, tetra-n-butylammonium fluoride (TBAF) to remove the 2xe2x80x2-OH alkylsilyl protecting groups, and gel purification and analysis of the deprotected RNA. Examples of chemical synthesis, deprotection, purification and analysis procedures for RNA are provided by Usman et al., 1987 J. Am. Chem. Soc., 109, 7845; Scaringe et al. Nucleic Acids Res. 1990, 18, 5433-5341; Perreault et al. Biochemistry 1991, 30 4020-4025; Slim and Gait Nucleic Acids Res. 1991, 19, 1183-1188. All the above noted references are all hereby incorporated by reference herein.
The deprotection process commonly involves the deprotection of the exocyclic amino protecting groups by NH4OH, which is time consuming (6-24 h) and inefficient. This step is then followed by treatment with TBAF to facilitate the removal of alkylsilyl protecting groups, which again is time consuming and not very effective in achieving efficient deprotection.
A recent modification of this two-step strategy for oligoribonucleotide deprotection has been reported by Wincott et al., (Nucleic Acids Res., 1995, 23, 2677-2784) and by Vinayak et al., (Nucleic Acids Symposium series, 1995. 33, 123-125). The optimized conditions make use of aqueous methylamine at 65xc2x0 C. for 15 minutes in place of the ammonium hydroxide cocktail to remove exocyclic amino protecting groups while the desilylation treatment needed to remove the 2xe2x80x2-OH alkylsilyl protecting groups utilizes a mixture of triethylamine trihydrogen fluoride (TEA.3HF), N-methyl-pyrrolidinone and triethylamine at 65xc2x0 C. for 90 minutes, thereby replacing tetrabutyl ammonium fluoride.
Stinchcomb et al., International PCT Publication No. WO 95/23225 describe a process for one pot deprotection of RNA. On page 73, it states that:
xe2x80x9cIn an attempt to minimize the time required for deprotection and to simplify the process of deprotection of RNA synthesized on a large scale, applicant describes a one pot deprotection protocol. According to this protocol, anhydrous methylamine is used in place of aqueous methyl amine. Base deprotection is carried out at 65xc2x0 C. for 15 minutes and the reaction is allowed to cool for 10 min. Deprotection of 2xe2x80x2-hydroxyl groups is then carried out in the same container for 90 minutes in a TEAxe2x80xa23HF reagent. The reaction is quenched with 16 mM TEAB solution.xe2x80x9d
This invention concerns a one-pot process for the deprotection of RNA molecules. This invention features a novel method for the removal of protecting groups from the nucleic acid base and 2xe2x80x2-OH groups, which accelerates the process for generating synthetic RNA in a high throughput manner (e.g., in a 96 well format).
Chemical synthesis of RNA is generally accomplished using a traditional column format on a RNA synthesizer where only one oligoribonucleotide is synthesized at a time. Simultaneous synthesis of more than one RNA molecule in a time efficient manner requires alternate methods to the traditional column format, such as synthesis in a 96 well plate format where up to 96 RNA molecules can be synthesized at the same time. To expedite this process of simultaneous synthesis of multiple RNA molecules, it is important to accelerate some of the time consuming processes such as the deprotection of RNA following synthesis (i.e. removal of base protecting group, such as the exocyclic amino protecting group and the phosphate protecting groups and the removal of 2xe2x80x2-OH protecting groups, such as the tButylDiMethylSilyl). In a preferred embodiment, the invention features a one-pot process for rapid deprotection of RNA.
Stinchcomb et al., supra described a one-pot protocol for RNA deprotection using anhydrous methylamine and triethylamine trihydrogen fluoride. This procedure involves the use of an anhydrous solution of base such as a 33% methylamine in absolute ethanol followed by neat triethylamine trihydrofluoride to effectively deprotect oligoribonucleotides in a one-pot fashion. However such a protocol may be cumbersome for deprotection of RNA synthesized on a plate format, such as a 96 well plate, because it may be necessary to separate the solid-support from the partially deprotected RNA prior to the 2xe2x80x2-hydroxyl deprotection. Also, since the methylamine solution used is anhydrous, it may be difficult to solubilize the negatively charged oligoribonucleotides obtained after basic treatment. So, in a first aspect the invention features the use of a 1:1 mixture of the ethanolic methylamine solution and a polar additive, such as dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), methanol, hexamethylphosphoramide (HMPA), 1-methyl-2-pyrrolidinone (NMP), 2-methoxyethyl ether (glyme) or the like. More specifically, dimethylsufoxide is used to partially deprotect oligoribonucleotides (FIG. 2). A comparison of the one pot and two pot deprotection methods are outlined and demonstrated in FIG. 3.
This invention also concerns a rapid (high through-put) deprotection of RNA in a 96-well plate format. More specifically rapid deprotection of enzymatic RNA molecules in greater than microgram quantities with high biological activity is featured. It has been determined that the recovery of enzymatically active RNA in high yield and quantity is dependent upon certain critical steps used during its deprotection.
In a preferred embodiment, the invention features a process for one-pot deprotection of RNA molecules comprising protecting groups, comprising the steps of: a) contacting the RNA with a mixture of anhydrous alkylamine (where alkyl can be branched or unbranched, ethyl, propyl or butyl and is preferably methyl, e.g., methylamine), trialkylamine (where alkyl can be branched or unbranched, methyl, propyl or butyl and is preferably ethyl, e.g., ethylamine) and dimethylsulfoxide, preferably in a 10:3:13, or 1:0.3:1 proportion at temperature 20-30xc2x0 C. for about 30-100 minutes, preferably 90 minutes, to remove the exocyclic amino (base) protecting groups and the phosphate protecting group (e.g., 2-cyanoethyl) (vs 4-20 h at 55-65xc2x0 C. using NH4OH/EtOH or NH3/EtOH, or 10-15 min at 65xc2x0 C. using 40% aqueous methylamine) under conditions suitable for partial deprotection of the RNA; b) contacting the partially deprotected RNA with anhydrous triethylaminexe2x80xa2hydrogen fluoride (3HFxe2x80xa2TEA) and heating at about 50-70xc2x0 C., preferably at 65xc2x0 C., for about 5-30 min, preferably 15 min to remove the 2xe2x80x2-hydroxyl protecting group (vs 8-24 h using TBAF, or TEAxe2x80xa23HF for 24 h (Gasparutto et al. Nucleic Acids Res. 1992, 20, 5159-5166) (Other alkylaminexe2x80xa2HF complexes may also be used, e.g. trimethylamine or diisopropylethylamine) under conditions suitable for the complete deprotection of the RNA. The reaction can then be quenched by using aqueous ammonium bicarbonate (1.4 M). Although some other buffers can be used to quench the desilylation reaction (i.e. triethylammonium bicarbonate, ammonium acetate), the ammonium bicarbonate buffer is perfectly suited to retain the 5xe2x80x2-O-dimethoxytrityl group at the 5xe2x80x2-end of the oligoribonucleotide thereby facilitating a reverse phase-based solid-phase extraction purification protocol.
By xe2x80x9cone-potxe2x80x9d deprotection is meant that the process of deprotection RNA is carried out in one container instead of multiple containers as in two-pot deprotection.
In another preferred embodiment, the invention features a process for one pot deprotection of RNA molecules comprising protecting groups, comprising the steps of: a) contacting the RNA with a mixture of anhydrous alkylamine (where alkyl can be branched or unbranched, ethyl, propyl or butyl and is preferably methyl, e.g., methylamine), and dimethylsulfoxide, preferably in a 1:1 proportion at 20-30xc2x0 C. temperature for about 30-100 minutes, preferably 90 minutes, to remove the exocyclic amino (base) protecting groups and the phosphate protecting group (e.g., 2-cyanoethyl) (vs 4-20 h at 55-65xc2x0 C. using NH4OH/EtOH or NH3/EtOH, or 10-15 min at 65xc2x0 C. 40% aqueous methylamine) under conditions suitable for partial deprotection of the RNA; b) contacting the partially deprotected RNA with anhydrous triethylaminexe2x80xa2hydrogen fluoride (3HFxe2x80xa2TEA) and heating at about 50-70xc2x0 C., preferably at 65xc2x0 C., for about 5-30 min, preferably 15 min to remove the 2xe2x80x2-hydroxyl protecting group (Other alkylaminexe2x80xa2HF complexes may also be used, e.g. trimethylamine or diisopropylethylamine) under conditions suitable for the complete deprotection of the RNA. The reaction can then be quenched by using aqueous ammonium bicarbonate (1.4 M). Although some other buffers can be used to quench the desilylation reaction (i.e. triethylammonium bicarbonate, ammonium acetate), the ammonium bicarbonate buffer is perfectly suited to retain the 5xe2x80x2-O-dimethoxytrityl group at the 5xe2x80x2-end of the oligoribonucleotide thereby facilitating a reverse phase-based solid-phase extraction purification protocol.
In another aspect the invention features a process for RNA deprotection where the exocyclic amino and phosphate deprotection reaction is performed with the ethanolic methylamine solution at room temperature for about 90 min or at 65xc2x0 C. for 15 min or at 45xc2x0 C. for 30 min or at 35xc2x0 C. for 60 min.
In a preferred embodiment, the process for deprotection of RNA of the present invention is used to deprotect a ribozyme synthesized using a column format as described in (Scaringe et al., supra; Wicott et al., supra).
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.